REVIEW

Chromatin as dynamic 10-nm fibers

Kazuhiro Maeshima & Ryosuke Imai & Sachiko Tamura &

Tadasu Nozaki

Received: 15 January 2014 /Revised: 18 March 2014 /Accepted: 20 March 2014 /Published online: 16 April 2014# The Author(s) 2014. This article is published with open access at Springerlink.com

Abstract Since Flemming described a nuclear substance inthe nineteenth century and named it “chromatin,” this sub-stance has fascinated biologists. What is the structure ofchromatin? DNA is wrapped around core histones, forminga nucleosome fiber (10-nm fiber). This fiber has long beenassumed to fold into a 30-nm chromatin fiber and subsequent-ly into helically folded larger fibers or radial loops. However,several recent studies, including our cryo-EM and X-ray scat-tering analyses, demonstrated that chromatin is composed ofirregularly folded 10-nm fibers, without 30-nm chromatinfibers, in interphase chromatin and mitotic chromosomes.This irregular folding implies a chromatin state that is physi-cally less constrained, which could be more dynamic com-pared with classical regular helical folding structures. Consis-tent with this, recently, we uncovered by single nucleosomeimaging large nucleosome fluctuations in living mammaliancells (∼50 nm/30 ms). Subsequent computational modelingsuggested that nucleosome fluctuation increases chromatinaccessibility, which is advantageous for many “targetsearching” biological processes such as transcriptional regu-lation. Therefore, this review provides a novel view on chro-matin structure in which chromatin consists of dynamic anddisordered 10-nm fibers.

Introduction

There are 60 trillion cells in the human body. Each cellcontains 2 m of genomic DNA in a small nucleus with anapproximately 10-μm diameter (a volume of only ∼100 fL to1 pL), and yet, it is able to search and read the information inits genomic DNA to execute diverse cellular functions. There-fore, it is important to understand how this long genomicDNA is organized in the nucleus. In the nineteenth century,W. Flemming described a nuclear substance that was clearlyvisible after staining with a basic dye using primitive lightmicroscopes and named it “chromatin.” This is now thoughtto be the basic unit of genomic DNA organization (Olins andOlins 2003). Since then, even before the discovery of thestructure of DNA (Watson and Crick 1953), chromatin hasattracted significant interest from biologists. In this reviewarticle, we assess the available data to provide a novel viewof chromatin in which “chromatin is a dynamic and disordered10-nm fiber.”

DNA and nucleosomes

Deoxyribonucleic acid (DNA) is a negatively charged poly-mer that produces electrostatic repulsion between adjacentDNA regions. Therefore, it would be difficult for a longDNA molecule alone to fold into a small space like thenucleus (Bloomfield 1996; Yoshikawa and Yoshikawa2002). To overcome this problem, the long, negativelycharged polymer is wrapped around a basic protein complexknown as a core histone octamer, which consists of the histoneproteins H2A, H2B, H3, and H4, to form a nucleosome(Fig. 1) (Olins and Olins 1974; Kornberg 1974; Woodcocket al. 1976). The structure of a nucleosome is well known atatomic resolution (1.9 Å) (Davey et al. 2002): 147 base pairs(bp) of DNA are wrapped in 1.7 left-handed superhelical turns

K. Maeshima :R. Imai : S. Tamura : T. NozakiBiological Macromolecules Laboratory, Structural Biology Center,National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan

K. Maeshima (*) :R. ImaiDepartment of Genetics, School of Life Science,Graduate University for Advanced Studies (Sokendai),Mishima, Shizuoka 411-8540, Japane-mail: kmaeshim@nig.ac.jp

T. NozakiInstitute for Advanced Biosciences, Keio University,Fujisawa 252-8520, Japan

Chromosoma (2014) 123:225–237DOI 10.1007/s00412-014-0460-2

around a histone octamer, whose surface is positively charged.Each nucleosome particle is connected by linker DNA (20–80 bp) to form repetitive motifs of ∼200 bp; this was describedoriginally to resemble “beads on a string” (Fig. 1) (Olins andOlins 2003). This nucleosome fiber is also known as the 10-nm fiber (Fig. 1). A single histone octamer in the nucleosomehas ∼220 positively charged lysine and arginine residues and∼74 negatively charged aspartic acid and glutamic acid resi-dues. There are also 400 negative charges in the phosphatebackbone of 200 bp of DNA. Because only about half of thenegative charges in the DNA are neutralized, the remainingcharge must be neutralized by other factors (e.g., linker his-tone H1, cations, and other positively charged molecules) forfurther folding.

Discovery of 30-nm chromatin fibers in vitro

In 1976, Finch and Klug first found, under transmissionelectron microscopy (EM), that purified nucleosome fibers(10-nm fibers) with linker histone H1 or Mg2+ ions werefolded into fibers with a diameter of 30 nm. They named these

fibers “30-nm chromatin fibers” (Figs. 1 and 2a, b) (Finch andKlug 1976). In their structural model of the 30-nm fiberscalled “solenoids,” consecutive nucleosomes are located ad-jacent to one another in the fiber and folded into a simple“one-start helix” (Fig. 2a). Subsequently, a second model ofthe “two-start helix” was proposed based on microscopicobservations of isolated nucleosomes (Fig. 2b) (Woodcocket al. 1984). The second model assumed that nucleosomeswere arranged in a zigzag manner, where a nucleosome in thefiber was bound to the second neighbor (Bassett et al. 2009)(Fig. 2b). In addition to these two famous structural models,many other structural variations of 30-nm chromatin fibershave been proposed (van Holde and Zlatanova 2007).

Fig. 1 Old and novel views of chromatin structure. A long DNA mole-cule with a diameter of ∼2 nm is wrapped around a core histone octamerand forms a nucleosome with a diameter of 11 nm (Alberts et al. 2007).The nucleosome has long been assumed to fold into 30-nm chromatinfibers (left) and subsequently into the higher order organization of inter-phase nuclei or mitotic chromosomes. The right panel shows the novelhypothesis of irregularly folded nucleosome fibers

Fig. 2 Two classical models of 30-nm chromatin fibers and higher orderchromatin structures. a One-start helix (solenoid), b two-start helix(zigzag). (Top) A scheme of the two different topologies of chromatin fibersis shown (Robinson and Rhodes 2006). Positions from the first (N1) to theeighth (N8) nucleosome are labeled. c Two classical higher order chromatinstructure models: the hierarchical helical folding model (Sedat andManuelidis 1978) and the radial loop model (Laemmli et al. 1978). In theradial loop model, many loop structures of the 30-nm fiber (red) wraparound the scaffold structure (gray) (Laemmli et al. 1978), which consistsof condensin and topoisomerase IIα (Maeshima and Laemmli 2003)

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Although 30-nm chromatin fibers have been studied exten-sively using various techniques, including biochemistry, bio-physics, X-ray crystallography, conventional EM, cryo-EM,and small-angle X-ray scattering (SAXS) (Finch and Klug1976; Woodcock et al. 1984; Widom and Klug 1985; Dorigoet al. 2004; Schalch et al. 2005; Robinson et al. 2006; Bordaset al. 1986; Langmore and Paulson 1983; Hansen 2002;Gilbert et al. 2004; Bystricky et al. 2004; Kruithof et al.2009), their definitive structure remains elusive (van Holdeand Zlatanova 2007; Tremethick 2007; for more recentreviews, see Grigoryev and Woodcock 2012; Ghirlando andFelsenfeld 2013; Bian and Belmont 2012). Defining a specificstructure for the 30-nm chromatin fibers may not be valid forseveral reasons. First, the Rhodes group suggested that thesolenoid or zigzag method of compaction was defined by thelength of the nucleosomal linker DNA (Routh et al. 2008).Second, Grigoryev et al. demonstrated that two-start zigzagand one-start solenoids could be present simultaneously in a30-nm chromatin fiber under certain conditions (Grigoryevet al. 2009; Grigoryev and Woodcock 2012). It should beemphasized that even a variation in linker DNA length of1 bp will correspond to a 36° rotation of one nucleosome withrespect to its neighbor and will cause significant structuralchanges in the fiber (van Holde and Zlatanova 2007).

Although the defined structure of 30-nm chromatin fibersremains unclear, it has long been assumed that the 10-nmnucleosome fibers form a 30-nm chromatin fiber and, subse-quently, the higher order chromatin structures of interphasenuclei and mitotic chromosomes. Several models have beenproposed to describe the structure of higher order chromatin.The “hierarchical helical folding model” suggests that a 30-nmchromatin fiber is folded progressively into larger fibers, includ-ing ∼100-nm and then ∼200-nm fibers, to form large interphasechromatin fibers (chromonema fibers) or mitotic chromosomes(Fig. 2c) (Sedat and Manuelidis 1978; Belmont et al. 1989;Belmont and Bruce 1994; for a review, see Horn and Peterson2002). In contrast, the “radial loop model” assumes that a 30-nmchromatin fiber folds into radially oriented loops to form mitoticchromosomes (Fig. 2c) (Paulson and Laemmli 1977; Laemmliet al. 1978; Marsden and Laemmli 1979).

Does the 30-nm chromatin fiber exist in vivo?The cryo-EM study

In 1986, the Dubochet group performed a pioneering study tovisualize native cellular structures using cryo-EM (Dubochetet al. 1986). Mammalian mitotic cells were frozen rapidly,sectioned, and observed directly under a cryo-EM with nochemical fixation or staining (cryo-EM of vitreous sections,CEMOVIS). The Dubochet group first observed “native”mammalian chromosomes in these sections. Mitotic chromo-somal regions were apparent because they were excluded

from electron-dense ribosomes and, therefore, were distin-guishable from the cytoplasmic regions, which are full ofribosomes (Dubochet et al. 1986; see also Maeshima andEltsov 2008). Surprisingly, the chromosomes had a homoge-neous, grainy texture with ∼11-nm spacing. No higher orderor periodic structures, including 30-nm fibers, were observed.This suggested that the basic structure of the chromosomewasa liquid-like compact aggregation of 10-nm, not 30-nm, nu-cleosome fibers (Dubochet et al. 1988).

Interphase chromatin has also been visualized using cryo-EM. Although the chromatin regions in interphase nuclei arenot as obvious as those in mitotic chromosomes because thereis no efficient chromatin marker in interphase nuclei, it wassuggested that interphase nuclei in most higher eukaryote cellsmight not contain 30-nm chromatin fibers (Dubochet andSartori Blanc 2001; Bouchet-Marquis et al. 2006; Fakan andvan Driel 2007). For example, typical heterochromatin re-gions in plant or mammalian nuclei resembled mitotic chro-mosomes by cryo-EM, forming a homogeneous texture with-out 30-nm structures (Bouchet-Marquis et al. 2006; Fakan andvan Driel 2007).

On the other hand, it is unclear whether the absence of 30-nmstructures in cryo-EM images truly demonstrates a lack of 30-nmchromatin fibers because when researchers capture cryo-EMimages, they use a technique called “defocusing” to producehigh-contrast images. This process results in artificial amplifica-tion or suppression of the signal intensity, which affects differentstructural features depending on the defocus value (contrasttransfer function [CTF] effect; for a review, see Frank 2006). Itis thus possible that the degree of defocusing needed to imagechromosomes or chromatin with high contrast prevents thevisualization of 30-nm chromatin fibers. To solve this problem,we collaborated with Eltsov, Frangakis, and Dubochet to com-pensate for the CTF effect by merging several images taken atdifferent levels of defocus into a single image (Conway andSteven 1999). Even after this correction, we were unable todetect 30-nm structures in the chromosomal areas. In addition,the detection of periodic structures in the chromosomal region bypower spectral (Fourier transform) analysis revealed a prominentpeak at 11 nm, but not at 30 nm. This cryo-EM study suggestedthat 30-nm chromatin fibers were essentially absent frommitoticchromosomes; therefore, we proposed that 10-nm nucleosomefibers exist in a highly disordered, interdigitated state similar to a“polymer melt” (Figs. 1 and 4) (Eltsov et al. 2008; Maeshimaet al. 2010a).

Small-angle X-ray scattering analyses revealed no 30-nmchromatin structures in interphase nuclei and mitoticchromosomes

Although our cryo-EM study did not detect any 30-nm struc-tures in mitotic chromosomes, it might be impossible to

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observe potential hierarchical regular structures becauseonly a small number of 50-nm sections were examined(Eltsov et al. 2008). Langmore and Paulson (Langmore andPaulson 1983; Paulson and Langmore 1983) detected a 30-nmstructure in interphase nuclei and mitotic chromosomes usingsmall-angle X-ray scattering (SAXS) analysis, which candetect bulky periodic structures in non-crystal materials insolution without chemical fixation or staining (Fig. 3a, b)(Roe 2000). Therefore, this study provided evidence for theexistence of 30-nm chromatin fibers in interphase chromatinand mitotic chromosomes (Langmore and Paulson 1983;Paulson and Langmore 1983). Because these findings wereinconsistent with the cryo-EM findings described above, weperformed a comprehensive investigation of the structure ofinterphase nuclei and mitotic chromosomes using SAXS andcryo-EM (Nishino et al. 2012; Joti et al. 2012; for a review, seeHansen 2012). Isolated human interphase nuclei andmitotic chromosomes were exposed to synchrotron X-raybeams (Fig. 3a). A typical scattering pattern of interphase

nuclei and mitotic chromosomes exhibited three peaks at30-, weakly at 11-, and 6-nm (Fig. 3c, left) (Nishino et al.2012; Joti et al. 2012). This was consistent with the previousfindings of Langmore and Paulson (1983), who suggestedthat the 6- and 11-nm peaks were derived from the face-to-face and edge-to-edge positioning of nucleosomes, respec-tively. They concluded that the 30-nm peak represented theside-by-side packaging of 30-nm chromatin fibers. However,this fails to explain why the 30-nm structures were notobserved in interphase chromatin and mitotic chromosomesusing cryo-EM.

To understand the nature of the 30-nm peak observed usingSAXS, isolated chromosomes were examined using cryo-EM(Nishino et al. 2012; Joti et al. 2012). Again, no 30-nmchromatin fibers were observed in chromosomes. However,the cryo-EM images revealed that the surface of the chromo-some was coated with electron-dense granules the size ofribosomes. Subsequent immunostaining and Western blottingconfirmed that the chromosome surface was contaminated

Fig. 3 Small angle X-rayscattering (SAXS) analysis ofchromatin structure. aExperimental design. Thechromosome pellet in a quartzcapillary tube was exposed tosynchrotron X-ray beams, and thescattering patterns were recordedusing the imaging plate (Nishinoet al. 2012). b When non-crystalmaterials were irradiated with X-rays, scattering at small anglesgenerally reflected periodicstructures. Images a and b werereproduced from Joti et al. (2012),with somemodifications. cUpperleft Typical SAXS patterns ofpurified mitotic HeLachromosome fractions. Threepeaks at ∼6, ∼11 (weak), and∼30 nm were detected (arrows).(Upper right) After the removalof ribosome aggregates, the 30-nm peak disappeared, whereas theother peaks remained. (Bottom) Amodel whereby the 30-nm peak inSAXS results from regularlyspaced ribosome aggregates andnot from the chromosomes.Image c was reproduced fromNishino et al. (2012), with somemodification

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with ribosomes. The ribosomes were stacked regularly at ∼30-nm intervals, which could explain the ∼30-nm peak observedusing SAXS. To test this hypothesis, we removed ribosomesfrom the surface of the chromosome by washing with anisotonic buffer-containing polyamine and EDTA (Lewis andLaemmli 1982) while maintaining the size and shape of thechromosomes and then analyzed mitotic chromosomes usingSAXS. Importantly, no 30-nm peaks were detected (Fig. 3c,right), but the 11- and 6-nm peaks resulting from the internalstructure of the nucleosomes remained (Fig. 3c, right). Simi-larly, when we examined the nuclei after ribosome removal,the 30-nm peak in the SAXS pattern disappeared (Joti et al.2012). These results suggested the absence of a 30-nm chro-matin fiber in interphase chromatin and mitotic chromosomes.

Next, we investigated the larger scale chromatin structureof interphase nuclei and mitotic chromosomes using a newlydeveloped apparatus for ultra-small-angle X-ray scattering(USAXS) (Nishino et al. 2009). Consistent with our previousobservations, there were no regular periodic structures be-tween ∼30- and 1,000-nm in interphase nuclei and mitoticchromosomes. This contradicts the hierarchical helical foldingmodel (Fig. 2c) (Nishino et al. 2012; Joti et al. 2012). Thescattering properties also suggested the existence of a scale-free structure or fractal nature up to ∼275-nm in interphasechromatin and ∼1,000-nm in mitotic chromosomes. This sug-gests that interphase and mitotic chromatin share the commonstructural features of up to ∼275 nm of condensed and irreg-ularly folded 10-nm nucleosome fibers without 30-nm struc-tures (discussed below). Taken together, the cryo-EM, SAXS,and USAXS data suggest that irregularly folded 10-nm nucle-osome fibers form the bulk structure of human interphasechromatin and mitotic chromosomes (Nishino et al. 2012;Joti et al. 2012). Nevertheless, it is possible that short stretchesof 30-nm fibers or other regularly folded hierarchies occur inhuman interphase chromatin and mitotic chromosomes.

Other evidence supporting the absence of 30-nmchromatin fibers

Dekker (2008) used the chromosome-conformation-capture(3C) technique to investigate the folding of a specific genomicDNA region within yeast cells. He measured the averagedistance between two loci in the genome by confocal micros-copy and the flexibility of the intervening chromatin fiber bythe 3C technique. In combination with polymer modeling, themass density of the chromatin fiber was determined. Hisconclusion was that yeast chromatin in a transcriptionallyactive domain did not form a compact 30-nm chromatin fiberbut rather was extended with a loose arrangement of 10-nmnucleosome fibers.

More recently, Bazett-Jones et al. used electron spectro-scopic imaging (ESI), a process that involves electron

microscopy with an energy filter. ESI makes it possible toperform phosphorus and nitrogen mapping in cells with highcontrast and resolution (Ahmed et al. 2009; Fussner et al.2011a, b). The signals from phosphorus and nitrogen, whichare the main components of DNA, may be used to assess thefolding of genomic DNA and can distinguish 10- from 30-nmfibers. They observed that pluripotent cells were characterizedby a highly dispersed mesh of 10-nm, but not 30-nm, fibers(Fussner et al. 2011a, b, 2012). In contrast, differentiated cellsform compact chromatin domains leave a large space in thenucleus that is devoid of DNA. Surprisingly, ESI combinedwith tomography methods revealed that condensed hetero-chromatin domains such as chromocenters consisted of 10-nm, rather than 30-nm, chromatin fibers (Fussner et al. 2012;for a review, see Quenet et al. 2012), consistent with theobservations using cryo-EM. Furthermore, Gan et al. investi-gated the picoplankton Ostreococcus tauri, the smallestknown free-living eukaryote, using cryo-EM tomography ofice sections and subsequent computational analysis (Gan et al.2013). They demonstrated thatO. tauri chromatin resembles adisordered assembly of nucleosomes without the 30-nm chro-matin structure compatible with the polymer melt model.Therefore, several lines of evidence suggest the absence ofregular 30-nm chromatin fibers in eukaryotic cells.

The absence of a 30-nm chromatin fiber in native chroma-tin may not be a surprise. Generally, native chromatin does nothave regularly spaced nucleosomes, so linker DNA lengthsvary. As pointed out by van Holde and Zlatanova (2007), eventhe addition of a single base to linker DNA changes therelative orientation of one nucleosome to the next by 36°.Unless nucleosome-nucleosome interactions are sufficient toovercome such variations, the formation of a regular chroma-tin fiber is impossible.

Why can 30-nm chromatin fibers be observed in vitro?

Although the near absence of 30-nm chromatin fibers ineukaryotic cells was suggested, these structures are shown inEM images in molecular biology textbooks. We propose thatmost 30-nm chromatin fibers in EM images are in vitro arti-facts caused by the low-salt buffer conditions. The formationof 30-nm chromatin fibers requires the selective binding ofnucleosomes, which are close neighbors on the DNA strand,via intra-fiber nucleosomal association (Fig. 4a). In low-saltbuffer conditions of <1 mM MgCl2 or <100 mM NaCl,nucleosomal fibers gently repel each other due to their nega-tive charges. This “isolation of nucleosome fibers” facilitatesthe intra-fiber nucleosomal association and the subsequentformation of stable 30-nm chromatin fibers (Fig. 4a, b). Inconventional EM imaging studies, these 30-nm fibers mightbe stabilized through chemical cross-linking (such as

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glutaraldehyde fixation) and then shrunk further after alcoholdehydration during sample preparation (Maeshima et al.2010b).

Polymer melt

It is important to assess chromatin structure under morephysiological salt conditions. Under these conditions, inter-fiber nucleosome interactions become increasingly dominant(Fig. 4a, b) (Maeshima et al. 2010a). Nucleosome fibers(10 nm) are forced to interdigitate, which interferes withthe formation and maintenance of 30-nm chromatin fibers.

This leads to the polymer melt (Maeshima et al. 2010a) or“self-oligomer” state (for a review, see Hansen 2002; Hansen2012) (Fig. 4a, b). In addition, inter-fiber nucleosome inter-actions increase significantly in the presence of >2 mMMg2+

ions (Zheng et al. 2005; Kan et al. 2009). However,it is important to note that the tail domain of histone H4mediates both 30 nm fiber formation (Dorigo et al. 2003)and inter-fiber nucleosome association (Kan et al. 2009).Consequently, inter-fiber nucleosome association can preventthe formation of 30-nm fibers by sequestering the H4 taildomain (Hansen 2012).

Presence of 30-nm chromatin fibers in specific cells

Although inter-fiber nucleosome associations supposedlydominate within cells, there are some specific cell typeswhose nuclei contain apparent 30-nm chromatin fibers, forexample chicken erythrocytes (Langmore and Schutt 1980;Woodcock 1994; Scheffer et al. 2011) and starfish sperm(Woodcock 1994; Scheffer et al. 2012). These cells areterminally differentiated, and so, transcription is almostsilenced. In mouse rod cells, a large dense heterochromatindomain is located in the center of the nucleus. The hetero-chromatin at the periphery of the domain is formed byclosely packed 30-nm fibers, whereas such fibers have notbeen detected in the centermost domain (Kizilyaprak et al.2010). We propose that the stable formation of 30-nm chro-matin fibers in these cells could play a role in robust genesilencing. Nevertheless, there must be a unique mechanismto facilitate intra-fiber nucleosome association in these spe-cific cells. One possibility is the presence of a larger numberof linker histones. Consistent with this, linker histones couldstabilize 30-nm chromatin fibers in vitro (for a review, seeHansen 2002). In chicken erythrocytes, linker histone H5 isdeposited in the chromatin fibers at ∼1.4 molecules/nucleosome (for a review, see Kowalski and Palyga 2011),whereas starfish sperm chromatin has ∼1.7 H1 molecules pernucleosome; in contrast, various somatic cells have 0.5−0.8 H1 per nucleosome (Woodcock et al. 2006). Specifichistone modifications or the binding of specific proteins mightalso be involved in the formation of stable 30-nm fibers forrobust gene silencing (Kowalski and Palyga 2011). Interest-ingly, the 30-nm fibers of peripheral heterochromatin inmouse rod photoreceptor cells contain acetylated histones,which are usually associated with active transcription andde-condensed (Kizilyaprak et al. 2010). Histone acetylationmight induce the isolation of nucleosome fibers (Fig. 4a)and subsequent intra-fiber nucleosomal association to formstable 30-nm chromatin fibers because histone acetylationseems to inhibit inter-fiber nucleosome association by repel-ling the increasingly negative charge of the nucleosomes(Szerlong et al. 2010; Liu et al. 2011).

Fig. 4 Polymer melt model. a Under low-salt conditions, nucleosomefibers could form 30-nm chromatin fibers via intra-fiber nucleosomeassociations. An increase in salt (cation) concentration results in inter-fiber nucleosomal contacts that interfere with intra-fiber nucleosomalassociations, leading to a polymer melt scenario. Note that in theseillustrations, we show a highly simplified two-dimensional nucleosomemodel. Arrows and dotted lines show repulsion forces and interactions,respectively. b During the melting process, the 30-nm chromatin fibersbecome irregularly folded nucleosome fibers

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Higher order interphase chromatin structures

As described above, several studies have demonstrated thatirregular folded 10-nm nucleosome fibers form the bulk struc-ture of interphase chromatin and mitotic chromosomes. Nev-ertheless, the higher order structure of chromatin must also beconsidered. Based on the available data, including studiesfrom our laboratory, we propose that interphase chromatinforms numerous condensed chromatin domains consisting ofirregularly folded 10-nm nucleosome fibers that resemble“chromatin liquid drops” (Fig. 5a) (Maeshima et al. 2010a;Joti et al. 2012). These domains can be considered to be dropsof viscous chromatin, which could be formed by the macro-molecular crowding effect (Asakura and Oosawa 1954) and

other specific proteins such as cohesin (Nasmyth and Haering2005; Hirano 2006) and/or condensin II (Ono et al. 2013;Thadani et al. 2012). Similar chromatin domains were pro-posed in the chromosome territory-interchromatin compart-ment (CT-IC) model (Cremer et al. 2000; Cremer and Cremer2001), where each CT is built from a series of interconnected1 Mb-sized chromatin domains. These domains were identi-fied originally using pulse labeling of DNA replication foci(Nakamura et al. 1986; Schermelleh et al. 2001; Berezneyet al. 2005; Albiez et al. 2006) that persisted stably in subse-quent cell generations (Jackson and Pombo 1998; Ma et al.1998; Zink et al. 1999). Several recent reports have used theHi-C and chromosome conformation capture carbon copy(5C) methods to investigate the three-dimensional architectureof genomic DNA within cells, and they have proposed thephysical packaging of genomic DNA. The DNA packing unitswere termed “topologically associating domains (TADs)”(Nora et al. 2012), “topological domains” (Dixon et al.2012), or “physical domains” (Sexton et al. 2012). Recentstudies have reported that TADs, which can be hundreds ofkilobases in size, were identified in fly, mouse, and humancells, suggesting that TADs could be universal building blocksof chromosomes. Loci located within TADs tend to interactfrequently with each other, but they interact much less fre-quently with loci located outside their domain.

What is the advantage of these condensed chromatin do-mains? A number of biological implications have been pro-posed for TADs (Nora et al. 2013). For instance, TADs werefound to correspond to lamin-associated chromatin domains(LADs) in nuclei (Guelen et al. 2008). Most DNA replicationdomains, where DNA replication takes place in a nearlysynchronous manner, overlap with multiple TADs (Rybaet al. 2010). Changes in timing during cell differentiationtypically involve TAD-sized regions. Regarding transcription-al regulation, enhancer-promoter interactions produced bylooping might be limited to elements located within the sameTAD (Shen et al. 2012). TADs might also be defined bygenetically encoded boundary elements (Nora et al. 2012).

In addition, we reported recently that condensed chromatinis more resistant to radiation damage than the decondensedform (Fig. 5b), presumably because condensed chromatin hasa lower level of reactive radical generation after ionizing irra-diation (Takata et al. 2013). The condensed state also protectsgenomic DNA from chemical attack. These findings suggestthat condensed chromatin domains play an important role inmaintaining genomic integrity (see also Falk et al. 2008).

Mitotic chromosome structure

Nucleosome fibers (10-nm) are somehow organized into mi-totic chromosomes. Condensins and topoisomerase IIα,which are essential for chromosome condensation, form an

Fig. 5 Higher order structure of interphase chromatin. a Condensedchromatin domains. Active chromatin regions are transcribed on thesurfaces of chromatin domains with transcriptional complexes (purplespheres) and RNA polymerase II (green spheres). NPC nuclear porecomplex, NE nuclear envelope. b (Left) Condensed chromatin is moreresistant to radiation damage or chemical attack. (Right) Reactive radicalsarising from the radiolysis of water molecules by irradiation can damagedecondensed chromatin; decondensed chromatin is also more accessibleto chemicals (labeled “Ch”)

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axis in the chromosome in various cell types (Hirano 2012;Thadani et al. 2012; Ohta et al. 2010; Belmont 2006;Maeshima and Eltsov 2008). Although it was claimed thatthe condensin axis was observed only in fixed and not livingcells (Thadani et al. 2012), we observed clear axial structuresof the condensin structures kleisin β- and γ-EGFP in chro-mosomes in living mammalian cells (Fig. 6a–c). Therefore,we hypothesized that condensins hold 10-nm nucleosomefibers around the chromosome center creating loops, as

proposed in the radial loop/scaffold model (Figs. 2c and 6d)(Laemmli et al. 1978; Maeshima and Eltsov 2008; Nishinoet al. 2012). Locally, nucleosome fibers are folded in anirregular manner towards the center of the chromosome(Fig. 6d) (Nishino et al. 2012). An immuno-EM study ofcondensins revealed a traceable condensin array near thecenter of chromosome cross-sections (Maeshima et al.2005), suggesting the oligomerization or self-assembly struc-ture of condensins, which capture nucleosome fibers.

Fig. 6 Mitotic chromosome structure. Axial localizations of condensins Iand II in mitotic chromosomes in live mammalian cells. For DNAstaining, DM (Indian Muntjac cells) cells stably expressing EGFP-Kleisin γ (condensin I) and EGFP-Kleisinβ (condensing II) were stainedwith Hoechst 33342. Live-cell imaging was performed using a DeltaVision microscope (applied precision). a Clear axial signals of EGFP-Kleisin γ in mitotic chromosomes are shown. b End-on-view of mitoticchromosomes. The upper panel shows DM cells expressing EGFP-Kleisin γ, whereas the lower panel shows DM cells expressing EGFP-Kleisin β stably. Restricted dot signals from two types of EGFP-Kleisin

in the cross-section of a chromosome body (DNA staining) are shown. cQuantitative data using line-profile analysis (blue line, DNA; red line,Kleisin signals) is shown. There is clear axial localization of condensins Iand II in mitotic chromosomes in live mammalian cells. d Chromosomesconsist of irregularly folded 10-nm nucleosome fibers. Condensins (blue)hold the nucleosome fibers (red) around the center of the chromosome.Locally, the nucleosome fibers are folded in an irregular or disorderedmanner, forming loop structures that collapse towards the center of thechromosome center (blue). The collapsed fiber (red) then forms a domain

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Condensins can also aggregate in the presence of DNA(Yoshimura et al. 2002; see also Hirano 2012). In our model,the orientation of nucleosome fibers in chromosomes is iso-tropic (Fig. 6d). This suggests that a specific locus of thegenome is randomly incorporated into a wide ranging, butnot reproducibly specific, region of the chromosome (Nishinoet al. 2012). This is consistent with data reported using fluo-rescent labeling of specific chromosomal sites (Strukov andBelmont 2009).

Naumova et al. (2013) recently performed 5C and Hi-Canalyses to understand the three-dimensional folding of geno-mic DNA in mitotic chromosomes. In human cells from G1 toS to G2 phase, they identified large chromatin structurescalled “chromosome compartments” (multi-megabases) andTADs (hundreds of kilobases), both of which were found inprevious studies (Lieberman-Aiden et al. 2009; Nora et al.2012; Dixon et al. 2012; Sexton et al. 2012). However, thesestructures were not found during mitosis; instead, they foundhomogenous folding of genomic DNA, which seems to beconsistent with our view that chromosomes consist of irregu-larly folded nucleosome fibers.

Using polymer simulations, they found that the obtaineddata for mitotic chromosomes are inconsistent with the classichierarchical helical folding model (Fig. 2c) and are, instead,best described by a linearly organized longitudinally com-pressed array of consecutive chromatin loops (Naumovaet al. 2013), which is essentially similar to the radialloop/scaffold model (Fig. 2c) (Laemmli et al. 1978; for areview, see Kleckner et al. 2013).

Dynamic 10-nm fibers in living mammalian cells

The original liquid chromatin model proposed by Dubochet(McDowall et al. 1986; Dubochet et al. 1988) and our polymermelt model (Eltsov et al. 2008; Maeshima et al. 2010a) bothimply a less physically constrained chromatin state and a morelocally dynamic state; the 10-nm nucleosome fibers fluctuatelocally. Therefore, we attempted to visualize local nucleosomefluctuation. Previous studies of chromatin dynamicsemployed very large chromatin regions such as the LacO arraythat encompasses 20−50 nucleosomes (Straight et al. 1996;Belmont et al. 1999; Heun et al. 2001; Vazquez et al. 2001;Chubb et al. 2002; Levi et al. 2005; Hajjoul et al. 2013). Themotion of these large regions in living mammalian cells wasmeasured by monitoring the movement of the GFP-LacIsignal bound to the LacO array at specific chromatin regions.

To observe and analyze more local nucleosome dynamics,we performed single nucleosome imaging in living mamma-lian cells (Fig. 7) (Hihara et al. 2012; Nozaki et al. 2013). Wefused histone H4 with photoactivatable (PA)-GFP andexpressed the fusion protein in mammalian cells at a verylow level (Fig. 7a). We then used an oblique illumination

microscope to illuminate a limited thin area within the cellfor single nucleosome imaging (Hihara et al. 2012; Nozakiet al. 2013; for principle, see Tokunaga et al. 2008). Generally,PA-GFP shows green fluorescence only after activation with a405-nm laser (Lippincott-Schwartz and Patterson 2009). Sur-prisingly, we observed that a small fraction of H4-PA-GFPand PA-GFP-H4 in the cells was activated spontaneouslywithout laser stimulation (Fig. 7a). Figure 7b shows a typicalsingle nucleosome image of a living mammalian cell. Eachbright dot in the nucleus represents a single H4-PA-GFP (PA-GFP-H4) within the single nucleosome. Strikingly, we ob-served significant nucleosome fluctuation (∼50 nm

Fig. 7 Single nucleosome imaging. a A small portion of PA-GFP-H4was activated spontaneously without laser activation and was used forsingle nucleosome imaging. b Single nucleosome image of a DM cell(Indian Muntjac cell) nucleus that expresses PA-GFP-H4. PA-GFP-H4 isobserved as a bright dot using oblique illumination microscopy. The dotswere fitted to an assumedGaussian point spread function to determine theprecise center of signals with higher resolution. Bar=5 μm. c Represen-tative three trajectories of fluorescently tagged single nucleosomes. dChromatin fluctuations as a basis for scanning genome information. Incells, nucleosome fibers (red spheres and lines) are folded irregularly. Thenucleosomes fluctuate, and these nucleosome dynamics facilitate chro-matin accessibility. The images were reproduced from (Hihara et al. 2012;Nozaki et al. 2013) with some modification

Chromosoma (2014) 123:225–237 233

movement/30 ms) in both interphase chromatin and mitoticchromosomes (Fig. 7c), which is likely caused by theBrownian motion (Hihara et al. 2012; Nozaki et al. 2013).Mean square displacement (MSD) plots, measuring the spatialextent of the random motion, and fitting to an anomalousdiffusion curve suggested a restricted nucleosome movement.The McNally group also published single nucleosome track-ing data using H2B-EGFP (Mazza et al. 2012), which appearsto be consistent with our single nucleosome tracking resultsusing PA-GFP-H4.

Local fluctuation of nucleosomes as a basis for scanninggenome information

Some computational modeling studies, including our own,have suggested that nucleosome fluctuations facilitate themobility of diffusing proteins in the chromatin environment(Fig. 7d) (Hihara et al. 2012; see also Wedemeier et al. 2009;Fritsch and Langowski 2011). Such nucleosome fluctuationsmay also contribute to the frequent exposure of genomic DNAsequences. Because both facilitating protein mobility andDNA exposure increase chromatin accessibility, these localdynamics may be advantageous in template-directed biologi-cal processes such as transcriptional regulation, DNA replica-tion, and DNA repair/recombination. Therefore, we proposethat the local fluctuation of nucleosomes forms the basis forscanning genome information (Fig. 7d).

We consider that nucleosome fluctuations are involved invarious cellular functions. Hinde et al. (2012) examined thechromatin dynamics in human ES cells based on signal inten-sity fluctuations of DAPI or H2B-EGFP, and they found thatthe intensity of the fluctuations in ES cells was drasticallyimpaired during differentiation, suggesting that such fluctua-tions correlate with pluripotency. A dynamic chromatin statemay be required for high transcriptional competency to main-tain pluripotency. Elucidation of the spatio-temporal regula-tion of nucleosome fluctuations would be an intriguing nextstep.

Conclusions

The traditional view of chromatin is changing from one ofstatic regular structures including 30-nm chromatin fibers to adynamic irregular folding structure of 10-nm nucleosomefibers. Although the term “irregular” or “disordered” mightgive the impression that the organization is functionally irrel-evant, the irregular folding results in less physical constraintand increased dynamism, increasing the accessibility of theDNA (Fig. 7d). This dynamic state may be essential forvarious genome functions, including transcription, replication,and DNA repair/recombination.

A new paper (Eltsov M, Sosnovski S, Olins AL, Olins DE:Chromosoma. 2014 Feb 26. [Epub ahead of print]) publishedafter this article went to press. The authors studied nuclearenvelope-limited chromatin sheets (ELCS) by cryo-EM. Theyfound that the 30-nm chromatin fibers could only be observedfollowing aldehyde fixation; none were seen in cryo-sections,suggesting that the 30-nm chromatin fibers in ELCS visualizedby conventional EM could be an artifact structure.

Acknowledgments We are grateful to Dr. Eltsov for stimulating dis-cussions and Drs. T. Sutani, M. Shimura, M. Sasai, K. Fujimura, S. Ide,C.-F. Kao, H. Masumoto, A. Sasaki, and H. Takata for critically readingthe manuscript. We would like to thank all our collaborators who con-tributed to our studies. We thank T. Cremer and I. Hiratani for the helpfuldiscussions and support. We must also apologize to our colleagues in thechromatin field for citing a limited number of their papers due to spaceconstraints. A grant-in-aid for a MEXT grant, JST CREST, YamadaScience Foundation, and Takeda Science Foundation supported thiswork. T. N. is a JSPS fellow.

Open Access This article is distributed under the terms of the CreativeCommons Attribution License which permits any use, distribution, andreproduction in any medium, provided the original author(s) and thesource are credited.

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